Inbred corn line wbb53

ABSTRACT

An inbred corn line, designated WBB53, is disclosed. The invention relates to the seeds of inbred corn line WBB53, to the plants and plant parts of inbred corn line WBB53 and to methods for producing a corn plant, either inbred or hybrid, by crossing the inbred line WBB53 with itself or another corn line. The invention further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other inbred corn lines derived from the inbred WBB53.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive corn inbred line,designated WBB53. All publications cited in this application are hereinincorporated by reference. There are numerous steps in the developmentof any novel, desirable plant germplasm. Plant breeding begins with theanalysis and definition of problems and weaknesses of the currentgermplasm, the establishment of program goals, and the definition ofspecific breeding objectives. The next step is selection of germplasmthat possess the traits to meet the program goals. The goal is tocombine in a single variety or hybrid an improved combination ofdesirable traits from the parental germplasm. These important traits mayinclude higher yield, resistance to diseases and insects, better stalksand roots, tolerance to drought and heat, reduction of grain moisture atharvest as well as better agronomic quality. With mechanical harvestingof many crops, uniformity of plant characteristics such as germinationand stand establishment, growth rate, maturity and plant and ear heightis important.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

The complexity of inheritance influences choice of breeding method.Backcross breeding is used to transfer one or a few favorable genes fora heritable trait into a desirable cultivar. This approach has been usedextensively for breeding disease-resistant cultivars, nevertheless, itis also suitable for the adjustment and selection of morphologicalcharacters, color characteristics and simply inherited quantitativecharacters such as earliness, plant height or seed size and shape.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s) for three or more years.The best lines are candidates for use as parents in new commercialcultivars; those still deficient in a few traits may be used as parentsto produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to twelve years from the time thefirst cross is made. Therefore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a focus on clear objectives.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of corn breeding is to develop new, unique and superior corninbred lines and hybrids. The breeder initially selects and crosses twoor more parental lines, followed by repeated self pollination or selfingand selection, producing many new genetic combinations. The breeder cantheoretically generate billions of different genetic combinations viacrossing, selfing and mutations.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.This unpredictability results in the expenditure of large research fundsto develop a superior new corn inbred line.

The development of commercial corn hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the crosses. Pedigree breeding and recurrent selection breedingmethods are used to develop inbred lines from breeding populations.Breeding programs combine desirable traits from two or more inbred linesor various broad-based sources into breeding pools from which inbredlines are developed by selfing and selection of desired phenotypes. Thenew inbreds are crossed with other inbred lines and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable cultivar or inbredline which is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding, John Wiley and Son, pp.115-161, 1960; Allard, 1960; Fehr, 1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock.

Hybrid corn seed is typically produced by a male sterility system or byincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. These and all patents referred toare incorporated by reference. In addition to these methods, Albertsenet al., U.S. Pat. No. 5,432,068 have developed a system of nuclear malesterility which includes: identifying a gene which is critical to malefertility, silencing this native gene which is critical to malefertility; removing the native promoter from the essential malefertility gene and replacing it with an inducible promoter; insertingthis genetically engineered gene back into the plant; and thus creatinga plant that is male sterile because the inducible promoter is not “on”resulting in the male fertility gene not being transcribed. Fertility isrestored by inducing, or turning “on”, the promoter, which in turnallows the gene that confers male fertility to be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran anti-sense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see,Fabinjanski, et al. EPO 89/0301053.8 publication no. 329,308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G. R., U.S. Pat. No. 4,936,904). Application ofthe gametocide, timing of the application, and genotype often limit theusefulness of the approach.

Corn is an important and valuable field crop. Thus, a continuing goal ofplant breeders is to develop stable, high yielding corn hybrids that areagronomically sound. The reasons for this goal are obviously to maximizethe amount of ears or kernels produced on the land used and to supplyfood for both humans and animals. To accomplish this goal, the cornbreeder must select and develop corn plants that have the traits thatresult in superior parental lines for producing hybrids.

SUMMARY OF THE INVENTION

According to the invention there is provided a novel inbred corn linedesignated WBB53. This invention thus relates to the seeds of inbredcorn line WBB53, to the plants or parts thereof of inbred corn lineWBB53, to plants or parts thereof having all the physiological andmorphological characteristics of inbred corn line WBB53 and to plants orparts thereof having all the physiological and morphologicalcharacteristics of inbred corn line WBB53 listed in Table 1 and asdetermined at the 5% significance level when grown in the sameenvironmental condition. Parts of the inbred corn plant of the presentinvention are also provided, such as e.g., pollen obtained from aninbred plant and an ovule of the inbred plant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of inbred corn plant WBB53. The tissue culturewill preferably be capable of regenerating plants having all thephysiological and morphological characteristics of the foregoing inbredcorn plant. Preferably, the cells of such tissue cultures will beembryos, meristematic cells, seeds, callus, pollen, leaves, anthers,roots, root tips, silk, flowers, kernels, ears, cobs, husks, stalks orthe like. Protoplasts produced from such tissue culture are alsoincluded in the present invention. The corn plants regenerated from thetissue cultures are also part of the invention.

Also included in this invention are methods for producing a corn plantproduced by crossing the inbred line WBB53 with itself or another cornline. When crossed with itself, i.e. crossed with another inbred lineWBB53 plant or self pollinated, the inbred line WBB53 will be conserved.When crossed with another, different corn line, an F1 hybrid seed isproduced. F1 hybrid seeds and plants produced by growing said hybridseeds are included in the present invention. A method for producing a F1hybrid corn seed comprising crossing inbred line WBB53 corn plant with adifferent corn plant and harvesting the resultant hybrid corn seed arealso part of the invention. The hybrid corn seed produced by the methodcomprising crossing inbred line WBB53 corn plant with a different cornplant and harvesting the resultant hybrid corn seed are included in theinvention, as are included the hybrid corn plant or parts thereof, seedsincluded, produced by growing said hybrid corn seed.

In another embodiment, this invention relates to a method for producingthe inbred line WBB53 from a collection of seeds, the collectioncontaining both inbred line WBB53 seeds and hybrid seeds having WBB53 asa parental line. Such a collection of seed might be a commercial bag ofseeds. Said method comprises planting the collection of seeds. Whenplanted, the collection of seeds will produce inbred line WBB53 plantsfrom inbred line WBB53 seeds and hybrid plant from hybrid seeds. Theplants having all the physiological and morphological characteristics ofcorn inbred line WBB53 or having a decreased vigor compared to the otherplants grown from the collection of seeds are identified as inbred lineWBB53 parent plants. Said decreased vigor is due to the inbreedingdepression effect and can be identified for example by a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small ear size, ear and kernel shape, ear color orother characteristics. As previously mentioned, if the inbred line WBB53is self-pollinated, the inbred line WBB53 will be preserved, therefore,the next step is controlling pollination of the inbred parent plants ina manner which preserves the homozygosity of said inbred line WBB53parent plant, and the final step is to harvest the resultant seed.

This invention also relates to methods for producing other inbred cornlines derived from inbred corn line WBB53 and to the inbred corn linesderived by the use of those methods.

In another aspect, the present invention provides transformed WBB53inbred corn line or parts thereof that have been transformed so that itsgenetic material contains one or more transgenes, preferably operablylinked to one or more regulatory elements. Also, the invention providesmethods for producing a corn plant containing in its genetic materialone or more transgenes, preferably operably linked to one or moreregulatory elements, by crossing transformed WBB53 inbred corn line witheither a second plant of another corn line, or a non-transformed cornplant of the inbred line WBB53, so that the genetic material of theprogeny that results from the cross contains the transgene(s),preferably operably linked to one or more regulatory elements. Theinvention also provides methods for producing a corn plant that containsin its genetic material one or more transgene(s), wherein the methodcomprises crossing the inbred corn line WBB53 with a second plant ofanother corn line which contains one or more transgene(s) operablylinked to one or more regulatory element(s) so that the genetic materialof the progeny that results from the cross contains the transgene(s)operably linked to one or more regulatory element(s). Transgenic cornplants, or parts thereof produced by the method are in the scope of thepresent invention.

More specifically, the invention comprises methods for producing malesterile corn plants, male fertile corn plants, herbicide resistant cornplants, insect resistant corn plants, disease resistant corn plants,water stress tolerant corn plants, or plants with modified, inparticular decreased, phytate content, plants with modified waxy and/oramylose starch content, plants with modified protein content, plantswith modified oil content or profile, plants with increaseddigestibility or plants with increased nutritional quality. Said methodscomprise transforming the inbred line WBB53 corn plant with nucleic acidmolecules that confer male sterility, male fertility, herbicideresistance, insect resistance, disease resistance, water stresstolerance, or that can modify the phytate, the waxy and/or amylosestarches, the protein or the oil contents, the digestibility or thenutritional qualities, respectively. The transformed corn plantsobtained from the provided methods, including male sterile corn plants,male fertile corn plants, herbicide resistant corn plants, insectresistant corn plants, disease resistant corn plants, water stresstolerant corn plants, plants with modified phytate, waxy and/or amylosestarches, protein or oil contents, plants with increased digestibilityand plants with increased nutritional quality are included in thepresent invention. For the present invention and the skilled artisan,disease is understood to be fungal disease, viral disease, bacterialdisease or other plant pathogenic diseases and disease resistant plantwill encompass plants resistant to fungal, viral, bacterial and otherplant pathogens.

Also included in the invention are methods for producing a corn plantcontaining in its genetic material one or more transgenes involved withfatty acid metabolism, carbohydrate metabolism, starch content such aswaxy starch or increased amylose starch. The transgenic corn plantsproduced by these methods are also part of the invention.

In another aspect, the present invention provides for methods ofintroducing one or more desired trait(s) into the corn line WBB53 andplants obtained from such methods. The desired trait(s) may be, but notexclusively, a single gene, preferably a dominant but also a recessiveallele. Preferably, the transferred gene or genes will confer suchtraits as male sterility, herbicide resistance, insect resistance,resistance for bacterial, fungal, or viral disease, male fertility,water stress tolerance, enhanced nutritional quality, modified waxycontent, modified amylose content, modified protein content, modifiedoil content, enhanced plant quality, enhanced digestibility andindustrial usage. The gene or genes may be naturally occurring maizegene(s) or transgene(s) introduced through genetic engineeringtechniques. The method for introducing the desired trait(s) ispreferably a backcrossing process making use of a series of backcrossesto the inbred corn line WBB53 during which the desired trait(s) ismaintained by selection.

When using a transgene, the trait is generally not incorporated intoeach newly developed line such as WBB53 by direct transformation.Rather, the more typical method used by breeders of ordinary skill inthe art to incorporate the transgene is to take a line already carryingthe transgene and to use such line as a donor line to transfer thetransgene into the newly developed line. The same would apply for anaturally occurring trait. The backcross breeding process comprises thefollowing steps: (a) crossing the inbred line WBB53 plants with plantsof another line that comprise the desired trait(s), (b) selecting the F₁progeny plants that have the desired trait(s); (c) crossing the selectedF₁ progeny plants with the inbred line WBB53 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have thedesired trait(s) and physiological and morphological characteristics ofcorn inbred line WBB53 to produce selected backcross progeny plants; and(e) repeating steps (c) and (d) one, two, three, four, five six, seven,eight, nine or more times in succession to produce selected, second,third, fourth, fifth, sixth, seventh, eighth, ninth or higher backcrossprogeny plants that comprise the desired trait(s) and all thephysiological and morphological characteristics of corn inbred lineWBB53 as listed in Table 1 and as determined at a 5% significance levelwhen grown in the same environmental conditions. The corn plantsproduced by the methods are also part of the invention. Backcrossingbreeding methods, well known to one skilled in the art of plant breedingwill be further developed in subsequent parts of the specification.

In a preferred embodiment, the present invention provides methods forincreasing and producing inbred line WBB53 seed, whether by crossing afirst inbred parent corn plant with a second inbred parent corn plantand harvesting the resultant corn seed, wherein both said first andsecond inbred corn plant are the inbred line WBB53 or by planting aninbred corn seed of the inbred corn line WBB53, growing an inbred lineWBB53 plant from said seed, controlling a self pollination of the plantwhere the pollen produced by the grown inbred line WBB53 plantpollinates the ovules produced by the very same inbred line WBB53 grownplant and harvesting the resultant seed.

The invention further provides methods for developing corn plants in acorn plant breeding program using plant breeding techniques includingrecurrent selection, backcrossing, pedigree breeding, molecular marker(Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms(RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily PrimedPolymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting(DAF), Sequence Characterized Amplified Regions (SCARs). AmplifiedFragment Length Polymorphisms (AFLPs), and Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, etc.) enhancedselection, genetic marker enhanced selection and transformation. Cornseeds, plants, and parts thereof produced by such breeding methods arealso part of the invention.

Definitions

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, allof which alleles relates to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein the morphological and physiological characteristicsof an inbred, determined at a 5% significance level when grown in thesame environmental conditions, are recovered in addition to the singlegene transferred into the inbred via the backcrossing technique or viagenetic engineering.

Daily heat unit value. The daily heat unit value is calculated asfollows: (the maximum daily temperature+the minimum daily temperature)/2minus 50. All temperatures are in degrees Fahrenheit. The maximumtemperature threshold is 86 degrees, if temperatures exceed this, 86 isused. The minimum temperature threshold is 50 degrees, if temperaturesgo below this, 50 is used.

HTU. HTU is the summation of the daily heat unit value calculated fromplanting to harvest.

Quantitative Trait Loci (QTL). Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Root Lodging. The root lodging is the percentage of plants that rootlodge; i.e., those that lean from the vertical axis at an approximate300 angle or greater would be counted as root lodged.

Stay Green. Stay green is the measure of plant health near the time ofblack layer formation (physiological maturity). A high score indicatesbetter late-season plant health.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties of plants)

Collection of seeds. In the context of the present invention acollection of seeds will be a grouping of seeds mainly containingsimilar kind of seeds, for example hybrid seeds having the inbred lineof the invention as a parental line, but that may also contain, mixedtogether with this first kind of seeds, a second, different kind ofseeds, of one of the inbred parent lines, for example the inbred line ofthe present invention. A commercial bag of hybrid seeds having theinbred line of the invention as a parental line and containing also theinbred line seeds of the invention would be, for example such acollection of seeds.

Decreased vigor. A plant having a decreased vigor in the presentinvention is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small ear size, ear and kernel shape, ear color orother characteristics.

Inbreeding depression. The inbreeding depression is the loss ofperformance of the inbreds due to the effect of inbreeding, i.e. due tothe mating of relatives or to self pollination. It increases thehomozygous recessive alleles leading to plants which are weaker andsmaller and having other less desirable traits.

Predicted RM. This trait for a hybrid, predicted relative maturity (RM),is based on the harvest moisture of the grain. The relative maturityrating is based on a known set of checks and utilizes conventionalmaturity such as the Comparative Relative Maturity Rating System or itssimilar, the Minnesota Relative Maturity Rating System.

MN RM. This represents the Minnesota Relative Maturity Rating (MN RM)for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

Yield (Quintals/Hectare). The yield is the actual yield of the grain atharvest adjusted to 15.5% moisture.

Moisture. The moisture is the actual percentage moisture of the grain atharvest.

GDU Silk. The GDU silk (=heat unit silk) is the number of growing degreeunits (GDU) or heat units required for an inbred line or hybrid to reachsilk emergence from the time of planting. Growing degree units arecalculated by the Barger Method, where the heat units for a 24-hourperiod are: GDU=((Max Temp+Min Temp)/2)−50 The highest maximum used is86° F. and the lowest minimum used is 50° F. For each hybrid, it takes acertain number of GDUs to reach various stages of plant development.GDUs are a way of measuring plant maturity.

Stalk Lodging. This is the percentage of plants that stalk lodge, i.e.,stalk breakage, as measured by either natural lodging or pushing thestalks determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

Plant Height. This is a measure of the height of the hybrid from theground to the tip of the tassel, and is measured in centimeters.

Ear Height. The ear height is a measure from the ground to the ear nodeattachment, and is measured in centimeters.

Dropped Ears. This is a measure of the number of dropped ears per plot,and represents the percentage of plants that dropped an ear prior toharvest.

Harvest Aspect. This is a visual rating given the day of harvest or theprevious day. Hybrids are rated 1 (poorest) to 9 (best) with poorerscores given for poor plant health, visible signs of fungal infection,poor plant intactness characterized by missing leaves, tassels, or othervegetative parts, or a combination of these traits.

Dry down. This is the rate at which a hybrid will reach acceptableharvest moisture

Pre-anthesis Brittle Snapping. This is a percentage of “snapped” plantsfollowing severe winds prior anthesis

Pre-anthesis Root Lodging. This is a percentage plants that root lodgeprior anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Post-anthesis Root Lodging. This is a percentage plants that root lodgeafter anthesis: plants that lean from the vertical axis at anapproximately 300 angle or greater.

Seedling Vigor. This is the vegetative growth after emergence at theseedling stage, approximately five leaves.

Seed quality. This is a visual rating assigned to the kernels of theinbred. Kernels are rated 1 (poorest) to 9 (best) with poorer scoresgiven for kernels that are very soft and shriveled with splitting of thepericarp visible and better scores for fully formed kernels.

Pollen shed. This is a visual rating assigned at flowering to describethe abundance of pollen produced by the anthers. Inbreds are rated 1(poorest) to 9 (best) with the best scores for inbreds with tassels thatshed more pollen during anthesis.

Plant habit. This is a visual assessment assigned during the latevegetative to early reproductive stages to characterize the plants leafhabit. It ranges from decumbent with leaves growing horizontally fromthe stalk to a very upright leaf habit, with leaves growing nearvertically from the stalk.

Plant intactness. This is a visual assessment assigned to a hybrid orinbred at or close to harvest to indicate the degree that the plant hassuffered disintegration through the growing season. Plants are ratedfrom 1 (poorest) to 9 (best) with poorer scores given for plants thathave more of their leaf blades missing.

Standability. A term referring to the how well a plant remains uprighttowards the end of the growing season. Plants with excessive stalkbreakage and/or root lodging would be considered to have poorstandability.

Late plant greenness. Similar to a stay green rating. This is a visualassessment given at around the dent stage but typically a few weeksbefore harvest to characterize the degree of greenness left in theleaves. Plants are rated from 1 (poorest) to 9 (best) with poorer scoresgiven for plants that have more non-green leaf tissue typically due toearly senescence or from disease.

Silking ability. This is a visual assessment given during flowering.Plants are rated on the amount and timing of silk production. Plants arerated from 1 (poorest) to 9 (best) with poorer scores given for plantsthat produce very little silks that are delayed past pollen shed.

GDU pollen. The number of heat units from planting until 50% of theplants in the hybrid are shedding pollen.

Plant Part. As used herein, the term “plant parts” includes leaves,stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs,husks, stalks, root tips, anthers, silk, tissue, cells and the like.

Plant Cell. Plant cell, as used herein includes plant cells whetherisolated, in tissue culture or incorporated in a plant or plant part.

DETAILED DESCRIPTION OF THE INVENTION

Inbred corn line WBB53 is a yellow dent corn which provides an excellentparental line in crosses for producing first generation (F₁) hybridcorn. Inbred corn line WBB53 is best adapted to the U.S. Corn Belt(Indiana, Illinois, Iowa and Nebraska) for seed production as it makesan outstanding female. Resulting hybrids are medium to late (108 to 118maturity days from planting to harvest) and are best suited for the corngrowing areas of Central and Southern Indiana and Illinois, Iowa andNebraska. Inbred corn line WBB53 shows good seedling vigor, good seedquality produced on white cobs, good husk cover, fair stay green,adequate to good pollen shed, dark green leaves and straight kernelrows. It has susceptibility to Gray Leaf Spot as a line but goodtolerance to Southern Corn Leaf Blight and to Stewart's Wilt.

WBB53 is similar to FR 1064 and flowers similarly, however there areseveral differences including the fact that WBB53 is more resistant toSouthern Corn Leaf Blight and to Stewart's Wilt but more susceptible toGray Leaf Spot. WBB53 also shows different silk color (salmon) comparedto FR 1064 (pale green).

WBB53 has a plant height of 223 cm with an average ear insertion of 86cm. WBB53 is a medium-late season inbred and is very well adapted foruse as a female in seed production. Average heat units to 50% pollenshed are approximately 1390 and to 50% silk are approximately 1398 asmeasured near Decatur, Ill. in 2003 and 2004.

WBB53 is an inbred line that contributes high yield potential, goodstalks and improved roots in hybrids. WBB53 hybrid combinations oftenresult in hybrids with girthy, well filled ears.

Some of the criteria used to select ears in various generations include:yield, percentage moisture, stalk quality, root quality, diseasetolerance, late plant greenness, late season plant intactness, tip fill,barrenness, ear shape and size, seed quality, ear height, pollenshedding ability, silking ability, and corn borer tolerance. During thedevelopment of the line, crosses were made to inbred testers for thepurpose of estimating the line's general and specific combining ability.The inbred was then evaluated further as a line and in numerous crossesby the Ft. Branch, Ind. USA corn research station and by other researchstations of AgReliant Genetics. The inbred has proven to have a goodcombining ability in numerous hybrid combinations for yield and/or otherdesirable agronomic traits.

The inbred line has shown uniformity and stability for the traits,within the limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in WBB53.

Inbred corn line WBB53 has the following morphologic and othercharacteristics (based primarily on data collected at Ft. Branch, Ind.and Decatur, Ill.). TABLE 1 VARIETY DESCRIPTION INFORMATION TABLE 1TYPE: Yellow Dent Corn REGION WHERE DEVELOPED:. Italy MATURITY: 113 DaysDays Heat Units From emergence to 50% of 64 1555 plants in silk: Fromemergence to 50% of 63 1529 plants in pollen: Heat Units: = GDU = ((MaxTemp + Min Temp)/2) − 50 PLANT: Plant Height to tassel tip: 223 cm(Standard Deviation = 4.47) Ear Height to base of top ear: 86 cm (8.94)Average Length of Top Ear Internode: 12.4 cm (1.67) Average number ofTillers: 6 (0.55) Average Number of Ears per Stalk: 1.8 (0.84)Anthocyanin of Brace Roots: 8 LEAF: Width of Ear Node Leaf: 9.6 cm(0.51) Length of Ear Node Leaf: 69.9 cm (2.10) Number of leaves abovetop ear: 6.0 (0.71) Leaf Angle (from 2nd Leaf above ear at anthesis toStalk above leaf): 12° (4.64) Leaf Color: Forrest Green Munsell Code7.5GY ¾ Leaf Sheath Pubescence (Rate on scale from 1 = none to 9 = likepeach fuzz: 1 Marginal Waves (Rate on scale from 1 = none to 9 = many):4.5 Longitudinal Creases (Rate on scale from 1 = none to 9 = many): 4TASSEL: Number of Lateral Branches: 4.8 (0.84) Branch Angle from CentralSpike: 45° Tassel Length (from top leaf collar to tassel top): 38.3 cm(0.58) Pollen Shed (Rate on scale from 0 = male sterile to 9 = heavyshed): 8 Anther Color: Dull yellow Munsell Code 5Y 8/8 Glume Color:Yellow green Munsell Code 2.5GY 8/6 Bar Glumes: No EAR: (Unhusked Data)Silk Color (3 days after emergence): Pink Munsell Code 5R 8/4 Fresh HuskColor (25 days after 50% silking): Olive Green Munsell Code 5GY 5/4 DryHusk Color (65 days after 50% silking): Olive Green Munsell Code 2.5GY5/4 Position of Ear: dropped Husk Tightness (Rate on scale from 1 = veryloose to 9 = very tight): 7 Husk Extension at harvest: full EAR: (HuskedEar Data) Ear Length: 13.7 cm (1.12) Ear Diameter at mid-point: 38.0 mm(1.23) Ear Weight: 93.6 gm (15.45) Number of Kernel Rows: 14.8 (1.10)Kernel Rows: N/A Row Alignment: straight Shank Length: 6.0 cm (0.85) EarTaper: N/A KERNEL: (Dried) Kernel Length: 10 mm (0.79) Kernel Width: 7.6mm (0.42) Kernel Thickness: 3.9 mm (0.42) Round Kernels (Shape Grade):30.4% Aleurone Color Pattern: N/A Aleurone Color: Dull Orange 10YR 8/8Hard Endosperm Color: Yellow Orange Munsell code 10YR 7/12 EndospermType: Dent Weight per 100 kernels (unsized sample): 20 gm (0.0) COB: CobDiameter at Mid-Point: 38.0 mm (1.23) Cob Color: White Munsell code N/AAGRONOMIC TRAITS: Stay Green (at 65 days after anthesis) (Rate on scalefrom 1 = worst to 9 = excellent): 1 % Dropped Ears (at 65 days afteranthesis): 0 % Pre-anthesis Brittle Snapping: 0 % Pre-anthesis RootLodging: 0 % Post-anthesis Root Lodging (at 65 days after anthesis): 0Yield of Inbred Per Se (at 12-13% grain moisture): Bu/Acre 95.2 atDecatur, Ill 8/04

Further Embodiments of the Invention

This invention is also directed to methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plantwherein either the first or second parent corn plant is an inbred cornplant of the line WBB53. Further, both first and second parent cornplants can come from the inbred corn line WBB53. When self-pollinated,or crossed with another inbred line WBB53 plant, the inbred line WBB53will be stable while when crossed with another, different corn line, anF1 hybrid seed is produced.

An inbred line is been produced through several cycles of selfpollination and shall therefore be considered as an homozygous line.

A hybrid variety is classically created through the fertilization of anovule from an inbred parental line by the pollen of another, differentinbred parental line. Due to the homozygous state of the inbred line,the produced gametes carry a copy of each parental chromosome. As boththe ovule and the pollen bring a copy of the arrangement andorganization of the genes present in the parental lines, the genome ofeach parental line is present in the resulting F₁ hybrid, theoreticallyin the arrangement and organization created by the plant breeder in theoriginal parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross is stable. The F₁ hybrid is then a combination ofphenotypic characteristics issued from two arrangement and organizationof genes, both created by a man skilled in the art through the breedingprocess.

Still further, this invention also is directed to methods for producingan inbred corn line WBB53-derived corn plant by crossing inbred cornline WBB53 with a second corn plant and growing the progeny seed, andrepeating the crossing and growing steps with the inbred corn lineWBB53-derived plant from 0 to 7 times. Thus, any such methods using theinbred corn line WBB53 are part of this invention: selfing, backcrosses,hybrid production, crosses to populations, and the like. All plantsproduced using inbred corn line WBB53 as a parent are within the scopeof this invention, including plants derived from inbred corn line WBB53.Advantageously, the inbred corn line is used in crosses with other,different, corn inbreds to produce first generation (F₁) corn hybridseeds and plants with superior characteristics.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which corn plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, kernels,seeds, ears, cobs, leaves, husks, stalks, roots, root tips, anthers,silk and the like.

Duncan, et al., Planta, 1985, 165:322-332 reflects that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both inbreds and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al., Plant Cell Reports, 1988, 7:262-265, reports severalmedia additions that enhance regenerability of callus of two inbredlines. Other published reports also indicated that “nontraditional”tissues are capable of producing somatic embryogenesis and plantregeneration. K. V. Rao et al., Maize Genetics Cooperation Newsletter,1986, 60:64-65, refers to somatic embryogenesis from glume calluscultures and B. V. Conger, et al., Plant Cell Reports, 1987, 6:345-347indicates somatic embryogenesis from the tissue cultures of corn leafsegments. Thus, it is clear from the literature that the state of theart is such that these methods of obtaining plants are “conventional” inthe sense that they are routinely used and have a very high rate ofsuccess.

Tissue culture of corn is described in European Patent Application,publication 160,390, incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes, Maize forBiological Research—Plant Molecular Biology Association,Charlottesville, Va., 1982, 367-372, and in Duncan et al., Planta, 1985,165:322-332. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce corn plants having thephysiological and morphological characteristics of inbred corn lineWBB53.

The utility of inbred corn line WBB53 also extends to crosses with otherspecies. Commonly, suitable species will be of the family Graminaceae,and especially of the genera Zea, Tripsacum, Croix, Schlerachne,Polytoca, Chionachne, and Trilobachne, of the tribe Maydeae. Potentiallysuitable for crosses with WBB53 may be the various varieties of grainsorghum, Sorghum bicolor (L.) Moench.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedcorn plants, using transformation methods as described below toincorporate transgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which, when placed under the control of plant regulatory signals,confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988); Jones et al., Mol. Gen. Genet., 210:86 (1987);Svab et al., Plant Mol. Biol. 14:197 (1990); Hille et al., Plant Mol.Biol. 7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate or bromoxynil. Comai et al.,Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618(1990) and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include beta-glucuronidase (GUS), beta-galactosidase,luciferase and chloramphenicol acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984). Another approach to the identification ofrelatively rare transformation events has been use of a gene thatencodes a dominant constitutive regulator of the Zea mays anthocyaninpigmentation pathway. Ludwig et al., Science 247:449 (1990).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells. Chalfieet al., Science 263:802 (1994). GFP and mutants of GFP may be used asscreenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain organs,such as leaves, roots, seeds and tissues such as fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred”. Promoters which initiate transcription only incertain tissue are referred to as “tissue-specific”. A “cell type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression incorn. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners Gatzet al., Mol. Gen. Genetics 243:32-38 (1994) or Tet repressor from Tn10(Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)). A particularlypreferred inducible promoter is a promoter that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter is the inducible promoter from a steroid hormone gene, thetranscriptional activity of which is induced by a glucocorticosteroidhormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression incorn or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in corn.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the ³⁵S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin corn. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in corn. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al.,Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129(1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834(1991), Gould et al., J. Cell. Biol.108:1657(1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, etal., Cell 39:499-509 (1984), Stiefel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is corn. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant inbred line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btalpha-endotoxin gene. Moreover, DNA molecules encoding alpha-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Pratt et al., Biochem. Biophys. Res. Comm. 163:1243(1989) (an allostatin is identified in Diploptera puntata). See alsoU.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-alpha-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-alpha-1,4-D-galacturonase. See Lamb et al., BioTechnology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., BioTechnology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT, bar, genes), and pyridinoxy or phenoxy propionicacids and cyclohexones (ACCase inhibitor-encoding genes). See, forexample, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses thenucleotide sequence of a form of EPSP which can confer glyphosateresistance. A DNA molecule encoding a mutant aroA gene can be obtainedunder ATCC accession number 39256, and the nucleotide sequence of themutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Europeanpatent application No. 0 333 033 to Kumada et al., and U.S. Pat. No.4,975,374 to Goodman et al., disclose nucleotide sequences of glutaminesynthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of a PAT gene is provided inEuropean application No. 0 242 246 to Leemans et al. DeGreef et al.,BioTechnology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for PAT activity. Exemplary ofgenes conferring resistance to phenoxy propionic acids and cyclohexones,such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knutzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992)

B. Increased resistance to high light stress such as photo-oxidativedamages, for example by transforming a plant with a gene coding for aprotein of the Early Light Induced Protein family (ELIP) as described inWO 03074713 in the name of Biogemma.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bact. 170:810 (1988)(nucleotide sequence of Streptococcus mutants fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,BioTechnology 10:292 (1992) (production of transgenic plants thatexpress Bacillus licheniformis alpha-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102:1045 (1993) (maize endosperm starch branching enzyme 11).

D. Increased resistance/tolerance to water stress or drought, forexample, by transforming a plant to create a plant having a modifiedcontent in ABA-Water-Stress-Ripening-Induced proteins (ARS proteins) asdescribed in WO 0183753 in the name of Biogemma, or by transforming aplant with a nucleotide sequence coding for a phosphoenolpyruvatecarboxylase as shown in WO02081714. The tolerance of corn to drought canalso be increased by an overexpression of phosphoenolpyruvatecarboxylase (PEPC-C4), obtained, for example from sorghum.

E. Increased content of cysteine and glutathione, useful in theregulation of sulfur compounds and plant resistance against variousstresses such as drought, heat or cold, by transforming a plant with agene coding for an Adenosine 5′ Phosphosulfate as shown in WO 0149855.

F. Increased nutritional quality, for example, by introducing a zeingene which genetic sequence has been modified so that its proteinsequence has an increase in lysine and proline. The increasednutritional quality can also be attained by introducing into the maizeplant an albumin 2S gene from sunflower that has been modified by theaddition of the KDEL peptide sequence to keep and accumulate the albuminprotein in the endoplasmic reticulum.

G. Decreased phytate content. 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

Methods for Corn Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and R1 plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and corn. Hiei etal., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 microm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299(1988), Klein et al., Biotechnology 6:559-563 (1988), Sanford, J. C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., BioTechnology 9:996 (1991). Alternatively,liposome and spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. D'Halluin et al., Plant Cell 4:1495-1505 (1992) andSpencer et al., Plant Mol. Biol. 24:51-61 (1994).

Following transformation of corn target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

When the term inbred corn plant is used in the context of the presentinvention, this also includes any inbred corn plant where one or moredesired trait has been introduced through backcrossing methods, whethersuch trait is a naturally occurring one or a transgenic one.Backcrossing methods can be used with the present invention to improveor introduce one or more characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene or the genes for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Fehr, 1987).

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to a second inbred (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a corn plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, generally determined at a 5% significance level when grown in thesame environmental conditions, in addition to the gene or genestransferred from the nonrecurrent parent. It should be noted that some,one, two, three or more, self-pollination and growing of a populationmight be included between two successive backcrosses. Indeed, anappropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving time, money andeffort for the breeder. A non limiting example of such a protocol wouldbe the following: a) the first generation F₁ produced by the cross ofthe recurrent parent A by the donor parent B is backcrossed to parent A,b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,fifth or more times to parent A to produce selected backcross progenyplants comprising the desired trait of parent B and physiological andmorphological characteristics of parent A. Step c) may or may not berepeated and included between the backcrosses of step d.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass not only visualinspection and simple crossing, but also follow up of thecharacteristic(s) through genetically associated markers and molecularassisted breeding tools. For example, selection of progeny containingthe transferred trait is done by direct selection, visual inspection fora trait associated with a dominant allele, while the selection ofprogeny for a trait that is transferred via a recessive allele, such asthe waxy starch characteristic, require selfing the progeny to determinewhich plant carry the recessive allele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, i.e. they may be naturally present in the non recurrentparent, examples of these traits include but are not limited to, malesterility, waxy starch, amylose starch, herbicide resistance, resistancefor bacterial, fungal, or viral disease, insect resistance, malefertility, water stress tolerance, enhanced nutritional quality,industrial usage, increased digestibility yield stability and yieldenhancement. An example of gene controlling resistance to rust funguswould be the Rp1D gene, which as other Rp resistance prevents P. sorghifrom producing spores. This Rp1D gene was usually preferred over theother Rp genes because it was widely effective against all races ofrust, but the emergence of new races lead to use other Rp genescomprising for example the Rp1E, Rp1G, Rp1I, Rp1K or “compound” geneswhich combine two or more Rp genes including Rp1GI, Rp1GDJ, etc. Thesegenes are generally inherited through the nucleus. Some known exceptionsto this are the genes for male sterility, some of which are inheritedcytoplasmically, but still act as single gene traits. Several of thesesingle gene traits are described in U.S. Pat. Nos. 5,777,196; 5,948,957and 5,969,212, the disclosures of which are specifically herebyincorporated by reference.

In 1981, the backcross method of breeding accounted for 17% of the totalbreeding effort for inbred line development in the United States,according to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of Allard (1960), published by JohnWiley & Sons, Inc, “Principles of Plant Breeding”). The method makes useof a series of backcrosses to the variety to be improved during whichthe character or the characters in which improvement is sought ismaintained by selection. At the end of the backcrossing, the gene orgenes being transferred, unlike all other genes, will be heterozygous.Selfing after the last backcross produces homozygosity for this genepair(s) and, coupled with selection, will result in a variety withexactly the adaptation, yielding ability and quality characteristics ofthe recurrent parent but superior to that parent in the particularcharacteristic(s) for which the improvement program was undertaken.Therefore, this method provides the plant breeder with a high degree ofgenetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because the same variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will bemodified only with regards to genes being transferred, which aremaintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from “Hope” wheat to “Bart” wheat and even pursuing thebackcrosses with the transfer of bunt resistance to create “Bart 38”,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in “CaliforniaCommon” alfalfa to create “Caliverde”. This new “Caliverde” varietyproduced through the backcross process is indistinguishable from“California Common” except for its resistance to the three nameddiseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,colour characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, “Calady”, has been produced by Jones andDavis. In dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. “Lady Wright”, along grain variety was used as the donor parent and “Coloro”, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety “Calady” was produced.

INDUSTRIAL APPLICABILITY

Corn is used as human food, livestock feed, and as raw material inindustry. The food uses of corn, in addition to human consumption ofcorn kernels, include both products of dry- and wet-milling industries.The principal products of corn dry milling are grits, meal and flour.The corn wet-milling industry can provide corn starch, corn syrups, anddextrose for food use. Corn oil is recovered from corn germ, which is aby-product of both dry- and wet-milling industries.

Corn, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs and poultry.

Industrial uses of corn include production of ethanol, corn starch inthe wet-milling industry and corn flour in the dry-milling industry. Theindustrial applications of corn starch and flour are based on functionalproperties, such as viscosity, film formation, adhesive properties, andability to suspend particles. The corn starch and flour have applicationin the paper and textile industries. Other industrial uses includeapplications in adhesives, building materials, foundry binders, laundrystarches, explosives, oil-well muds and other mining applications.

Plant parts other than the grain of corn are also used in industry, forexample: stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred corn line WBB53, the plant produced from the inbredseed, the hybrid corn plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid corn plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

Tables

In the tables that follow, the traits and characteristics of hybridcombination having inbred corn line WBB53 as a parental line are givencompared to other hybrids. The data collected are presented for keycharacteristics and traits. The field tests have been made at numerouslocations, with one or two replications per location. Information aboutthese hybrids, as compared to the check hybrids, is presented.

The first pedigree listed in the comparison group is the hybrid(s)containing WBB53. MON810 is the designation given by the MonsantoCompany (St. Louis, Mo.) for the transgenic event that, when expressedin maize, produces an endotoxin that is efficacious against the Europeancorn borer, Ostrinia nubilalis, and certain other Lepidopteran larvae.MON863 is the designation given by the Monsanto Company (St. Louis, Mo.)for the transgenic event that, when expressed in maize, produces anendotoxin that is efficacious against the western corn rootworm,Diabrotica virgifera, and certain other Coleopteran larvae. Informationfor each pedigree includes:

1. Mean yield in bushels/acre adjusted to 15% moisture of the hybridacross all locations is shown in the column under the heading MeanYield.

2. A mean for the percentage moisture for the hybrid across alllocations is shown in the column under the heading % Moist.

3. A mean of the percentage of plants with stalk lodging across alllocations is shown in the column under the heading % SL.

4. A mean of the percentage of plants with root lodging across alllocations is shown in the column under the heading % RL.

5. A mean of the stay green from 1 (poor) to 9 (good) is shown in thecolumn under the heading Stay Green.

6. Test weight is the grain density as measured in pounds per bushel andis shown in the column under the heading Test Weight.

7. A mean of ear height in cm is shown in the column under the headingEar Height.

8. A mean of plant height in cm is shown in the column under the headingPlant Height.

9. A mean of harvest aspect from 1 (poor) to 9 (good) is shown in thecolumn under the heading Harvest Aspect. TABLE 2 Overall Comparison:2003/8 Locations Mean % % % Test Ear Plant Harvest Pedigree Yield MoistSL RL Weight Height Height Aspect WBB53 × LH287MON810 179.5 19.1 5.0 1.754.0 110 300 5.4 WBB53 × LH287 171.1 19.0 2.9 4.4 54.0 132 295 4.6 At 8Locations As Compared to: P33P67 172.4 21.6 3.5 14.7 56.0 140 305 7.9RBO1 × LH287 171.8 16.9 4.2 10.9 54.2 97 280 4.8 RBO1 × LH287MON810170.7 17.6 4.6 1.5 53.6 102 285 4.6 KW4773 × LH287 162.2 20.3 4.1 8.952.0 125 295 6.2 LHC33 × LH287MON810 158.3 18.1 5.1 14.0 54.3 117 2955.3 LHC33 × LH287 154.7 17.3 3.8 10.7 54.3 102 290 5.0 P34B23 151.7 18.51.5 7.0 56.6 120 297 6.0

TABLE 3 Overall Comparisons: 2003/34 Locations Mean % % % Test PedigreeYield Moist SL RL Weight WBB53 × LH287MON810 195.6 18.6 5.9 5.7 55.1 At34 Locations As Compared to: P33P67 202.3 19.1 2.0 9.0 58.0 LH287MON810× RBO1 199.3 17.3 4.6 4.5 55.9 KW4773 × LH287MON810 197.8 19.2 4.1 15.554.4 KW4773 × LH287 194.2 18.8 5.1 16.5 54.3 LH287 × RBO1 191.9 16.8 1.42.4 55.7 LHC33 × LH287MON810 191.8 17.3 3.3 10.3 55.7 DKC6215 187.7 18.15.8 8.7 56.5

TABLE 4 Overall Comparisons: 2003/26 Locations Mean % % % Test Ear PlantStay Harvest Pedigree Yield Moist SL RL Weight Height Height GreenAspect WBB53 × LH283MON810 179.7 22.6 2.8 4.2 54.7 109 266 7.3 6.6 WBB53× LH287 174.2 21.1 4.4 2.2 52.8 114 283 4.5 5.1 WBB53 × LH283 167.3 21.95.2 2.4 54.6 114 268 6.5 5.5 At 26 Locations As Compared to: RBO1 ×LH287MON810 181.9 19.4 4.3 1.6 53.2 100 275 4.6 5.5 P33P67 181.6 22.26.4 2.3 55.8 125 302 5.6 6.4 LHC33 × LH283MON810 180.7 20.9 3.6 6.4 55.2113 276 7.2 6.7 KW4773 × LH287MON810 180.2 21.6 6.1 9.7 51.6 107 282 5.66.2 LHC33 × LH287MON810 178.3 20.4 5.9 5.8 53.3 107 288 4.7 5.8 KW4773 ×LH287 174.7 21.3 6.2 7.1 51.9 103 276 5.7 5.5 P31G98 174.3 22.5 6.7 1.653.9 135 307 6.2 5.8 LHC33 × LH283 171.4 20.7 7.1 1.2 55.2 113 271 6.65.7

TABLE 5 Overall Comparisons: 2003/27 Locations Mean % % % Test Ear PlantStay Harvest Pedigree Yield Moist SL RL Weight Height Height GreenAspect WBB53 × LH287MON810 183.4 21.2 6.8 5.1 52.8 115 287 5.2 5.4 At 27Locations As Compared to: RBO1 × LH287MON810 190.4 19.2 3.3 2.0 53.4 93279 4.5 5.8 P33P67 187.9 22.5 6.9 5.2 55.5 125 297 5.8 5.9 KW4773 ×LH287MON810 184.0 21.3 4.6 9.1 51.7 99 280 6.5 5.9 LHC33 × LH287 182.018.9 6.5 4.4 53.7 103 280 4.9 5.1 P34M95 180.7 18.7 2.8 1.8 55.4 99 2865.3 5.8 LH245MON810 × LH287 178.3 22.5 4.6 7.2 53.2 108 292 5.2 5.5LHC33 × LH287MON810 172.1 19.2 6.1 9.2 53.2 98 277 4.2 5.3

TABLE 6 Overall Comparisons: 2004/28 Locations Mean % % % Test Ear PlantHarvest Pedigree Yield Moist SL RL Weight Height Height Aspect WBB53 ×LH279 202.7 17.2 1.3 1.4 56.9 120 287 5.6 At 28 Locations As Comparedto: RBO1 × LH287MON810 207.4 17.2 0.4 3.1 56.8 102 291 5.9 P33D31 194.920.3 0.4 1.1 57.2 121 301 6.4 P33P67 194.8 20.2 2.0 2.2 57.7 133 314 6.3LHC33 × LH283 183.0 18.3 2.8 8.7 57.9 122 300 5.8

TABLE 7 Overall Comparisons: 2004/28 Locations Mean % % % Test Ear PlantStay Harvest Pedigree Yield Moist SL RL Weight Height Height GreenAspect WBB53 × LH287MON810 194.5 17.6 1.8 4.0 56.7 120 308 4.6 5.4 WBB53× LH259 188.9 19.9 1.7 4.6 56.8 112 278 5.7 5.2 WBB53 × LH283MON810180.9 18.9 2.5 5.5 57.6 117 280 6.6 5.7 At 27 Locations As Compared to:KW4773 × LH287 192.5 18.6 1.5 14.6 55.3 116 293 5.5 5.6 P33P67 187.519.1 3.4 6.3 58.2 130 310 5.7 5.7 P31A13 187.0 22.7 2.4 2.6 56.4 124 3126.1 5.9 LHC33 × LH259 180.2 19.0 1.5 5.6 56.9 112 290 5.7 5.3 LHC33 ×LH283 171.7 17.7 3.1 4.0 57.9 128 295 6.7 5.4

TABLE 8 Overall Comparisons: 2004/13 Locations Mean % % % Test StayHarvest Pedigree Yield Moist SL RL Weight Green Aspect WBB53 × LH279194.0 21.5 1.4 0.1 55.2 6.5 5.8 WBB53 × 188.8 21.8 1.3 0.6 55.2 6.8 5.8LH279MON863 WBB53 × 188.2 21.9 0.6 0.9 55.6 6.8 5.8 LH277MON810 WBB53 ×179.0 20.6 0.7 0.4 56.5 6.0 6.5 LH295MON810 At 13 Locations As Comparedto: LHC33 × 187.2 20.4 0.6 5.6 56.0 7.3 5.8 LH279MON863 P34H31 185.423.3 0.7 0.9 56.0 6.1 5.8 RBO1 × 182.4 21.3 0.0 0.2 55.6 6.3 6.3LH185MON810 LHC33 × LH279 180.3 20.6 1.3 1.2 55.7 7.3 5.2 RBO1 × 176.518.7 0.1 7.7 56.7 6.0 5.6 LH295MON810 P36R11 171.2 19.1 0.4 0.6 56.7 6.85.9

TABLE 9 Overall Comparisons: 2004/13 Locations Mean % % % Test HarvestPedigree Yield Moist SL RL Weight Aspect WBB53 × 210.7 17.1 3.7 12.057.2 4.8 LH287MON863 WBB53 × 210.2 17.9 0.9 4.1 56.7 5.2 LH287MON810WBB53 × 203.7 20.3 1.2 5.4 57.0 5.2 KW7641MON810 WBB53 × LH324 201.319.1 1.9 1.2 57.4 5.7 WBB53 × LH283 198.1 19.0 1.4 2.7 57.8 5.3 MON810At 13 Locations As Compared to: LHC34MON810 × LH324 219.6 18.1 1.1 3.057.7 6.9 P33N09 216.3 19.0 1.7 2.0 58.7 6.6 LH245 × 207.6 17.4 0.6 3.056.5 4.8 LH287MON810 RBO1 × 207.2 16.7 0.0 0.0 57.1 5.7 LH287MON810KW4773 × 207.1 18.9 0.2 3.7 55.3 6.1 LH287MON810 P33P67 206.5 19.7 3.70.7 58.2 5.3 LH332 × LH324 205.2 17.5 1.9 7.3 57.3 5.7 LHC33 × 196.217.3 0.5 7.8 57.3 4.9 LH287MON810 LHC33 × 192.9 16.5 3.2 13.5 57.9 4.8LH287MON863 LHC33 × 191.8 18.2 0.8 7.1 58.6 6.4 LH283MON810 RBO1 × LH324189.3 17.2 0.6 0.6 57.5 6.0

Deposit Information

A deposit of the inbred corn seed of this invention is maintained byAgReliant Genetics 4640 East State Road 32, Lebanon, Ind. 46052. Accessto this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patent andTrademarks to be entitled thereto under 37 CRF 1.14 and 35 USC 122. Uponallowance of any claims in this application, all restrictions on theavailability to the public of the variety will be irrevocably removed byaffording access to a deposit of at least 2,500 seeds of the samevariety with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110 or National Collections ofIndustrial, Food and Marine Bacteria (NCIMB), 23 St Machar Drive,Aberdeen, Scotland, AB24 3RY, United Kingdom.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somaclonal variants, variant individuals selected from large populationsof the plants of the instant inbred and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

1. A seed of corn inbred line designated WBB53, wherein a representativesample of seed of said line was deposited under NCIMB No.
 41356. 2. Acorn plant, or a part thereof, produced by growing the seed of claim 1.3. A corn plant, or a part thereof, having all the physiological andmorphological characteristics of the inbred line WBB53, wherein arepresentative sample of seed of said line was deposited under NCIMB No.41356.
 4. A tissue culture of cells produced from the plant of claim 2,wherein said cells of the tissue culture are produced from a plant partselected from the group consisting of leaves, pollen, embryos, roots,root tips, anthers, silks, flowers, kernels, ears, cobs, husks, seedsand stalks.
 5. A corn plant regenerated from the tissue culture of claim4, wherein the regenerated plant has all the morphological andphysiological characteristics of inbred line WBB53, wherein arepresentative sample of seed of said line was deposited under NCIMB No.41356.
 6. A method for producing a hybrid corn seed wherein the methodcomprises crossing the plant of claim 2 with a different corn plant andharvesting the resultant hybrid corn seed. 7.-14. (canceled)
 15. Amethod of producing a corn plant with waxy starch or increased amylosestarch wherein the method comprises transforming the corn plant of claim2 with a transgene that modifies the carbohydrate metabolism.
 16. A cornplant produced by the method of claim
 15. 17. A method of introducing adesired trait into corn inbred line WBB53 wherein the method comprises:(a) crossing the inbred line WBB53 plants grown from the inbred lineWBB53 seed, wherein a representative sample of seed of said line wasdeposited under NCIMB No. 41356, with plants of another corn line thatcomprise a desired trait to produce progeny plants, wherein the desiredtrait is selected from the group consisting of male sterility, malefertility, herbicide resistance, insect resistance, disease resistance,waxy starch, water stress tolerance, increased amylose starch andincreased digestibility; (b) selecting one or more progeny plants thathave the desired trait to produce selected progeny plants; (c) crossingthe selected progeny plants with the inbred line WBB53 plants to producebackcross progeny plants; (d) selecting for backcross progeny plantsthat have the desired trait and physiological and morphologicalcharacteristics of corn inbred line WBB53 listed in Table 1 to produceselected backcross progeny plants; and (e) repeating steps (c) and (d)three or more times in succession to produce selected fourth or higherbackcross progeny plants that comprise the desired trait and all of thephysiological and morphological characteristics of corn inbred lineWBB53 as listed in Table
 1. 18. A corn plant produced by the method ofclaim 17, wherein the plant has the desired trait and all of thephysiological and morphological characteristics of corn inbred lineWBB53 as listed in Table
 1. 19. A method for producing inbred line WBB53seed, wherein a representative sample of seed of said line was depositedunder NCIMB No. 41356, wherein the method comprises crossing a firstinbred parent corn plant with a second inbred parent corn plant andharvesting the resultant corn seed, wherein both said first and secondinbred corn plant are the corn plant of claim
 3. 20. A method forproducing inbred line WBB53 seed, wherein a representative sample ofseed of said line was deposited under NCIMB No. 41356, wherein themethod comprises: a) planting an inbred corn seed of claim 1; b) growinga plant from said seed; c) controlling pollination in a manner such thatthe pollen produced by the plant pollinates the ovules produced by theplant; and d) harvesting the resultant seed.
 21. A method for producinga male sterile corn plant wherein the method comprises transforming thecorn plant of claim 2 with a nucleic acid molecule that confers malesterility.
 22. A male sterile corn plant produced by the method of claim21.
 23. A method of producing an herbicide resistant corn plantcomprising transforming the corn plant of claim 2 with a transgene thatconfers herbicide resistance.
 24. An herbicide resistant corn plantproduced by the method of claim
 23. 25. The corn plant of claim 24,wherein the transgene confers resistance to an herbicide selected fromthe group consisting of imidazolinone, sulfonylurea, glyphosate,glufosinate, L-phosphinothricin, triazine and benzonitrile.
 26. A methodof producing an insect resistant corn plant wherein the method comprisestransforming the corn plant of claim 2 with a transgene that confersinsect resistance.
 27. An insect resistant corn plant produced by themethod of claim
 26. 28. The corn plant of claim 27, wherein thetransgene encodes a Bacillus thuringiensis endotoxin.
 29. A method ofproducing a disease resistant corn plant wherein the method comprisestransforming the corn plant of claim 2 with a transgene that confersdisease resistance.
 30. A disease resistant corn plant produced by themethod of claim
 29. 31. The corn plant of claim 18, wherein the desiredtrait is male sterility and the trait is conferred by a nucleic acidmolecule.
 32. The corn plant of claim 18, wherein the desired trait iswater stress tolerance.
 33. The corn plant of claim 18, wherein thedesired trait is increased digestibility.
 34. Hybrid corn seeddesignated WBB53*LH287 having inbred line WBB53 or a transgenic inbredline WBB53 as a parental line, wherein a representative sample of seedof WBB53 was deposited under NCIMB No. 41356, and having inbred lineLH287 or a transgenic inbred line LH287 as a parental line, wherein arepresentative sample of seed of LH287 was deposited under ATCCAccession No. PTA-1174, wherein the transgenic inbred line WBB53 orLH287 comprises a transgene that confers a trait selected from the groupconsisting of disease resistance, male sterility, herbicide resistance,and insect resistance.
 35. Hybrid corn seed designated WBB53*LH279having inbred line WBB53 or a transgenic inbred line WBB53 as a parentalline, wherein a representative sample of seed of WBB53 was depositedunder NCIMB No. 41356, and having inbred line LH279 or a transgenicinbred line LH279 as a parental line, wherein a representative sample ofseed of LH279 was deposited under ATCC Accession No. PTA-1172, whereinthe transgenic inbred line WBB53 or LH279 comprises a transgene thatconfers a trait selected from the group consisting of diseaseresistance, male sterility, herbicide resistance, and insect resistance.